Leishmania are obligate intracellular protozoan pathogens of humans. Within infected patients, various species of this organism inhabit and destroy macrophages within the skin or internal organs (i.e., spleen, liver and bone marrow). Thus, they cause either ulcerative, non-healing, disfiguring malignant skin lesions (e.g. L. mexicana) or degenerative and most often fatal visceral disease (e.g. L. donovani). According to World Health Organization estimates, these diseases afflict over 12 million patients annually in the Tropics and Neo-tropics worldwide. Our studies are aimed at defining the mechanisms involved in the pathophysiology of these organisms. In that regard, the basic cell, molecular and developmental biology of Leishmania and related trypanosomatid protozoa are investigated toward identifying and characterizing parasite molecules which are essential for the survival of these human pathogens. How these parasites are able to survive, access nutrients, multiply and differentiate within their insect vector and mammalian hosts are questions central to understanding the basic parasitic nature and evolutionary adaptations of these organisms. Since these parasites interact directly with their hosts, knowledge of the composition and functions of their surface membrane and other functional proteins seems essential. To that end, unique parasite surface membrane, secreted enzymes and regulatory proteins are identified and biochemically characterized to determine their functional roles in the survival of these organisms. Further, the genes encoding such proteins are being isolated and characterized for the first time, toward defining their expression and regulation during course of parasite growth, differentiation and development. For example, we have identified and characterized the genes that encode several unique L. donovani tartrate-sensitive, histidine, secretory-acid phosphatase enzymes (LdSAcPs). Recently, we showed using combined biochemical and molecular approaches that this family of enzymes was functionally conserved among all pathogenic species of Leishmania examined. This suggested that they must play significant functional roles in the growth, development and survival of all members of this important group of human pathogens. In order to function within their hosts, these LdSAcPs would have to be generally resistant to endogenous host proteases. In that regard, recent results showed that the LdSAcPs were virtually totally resistant to hydrolytic-degradation by various different serine-, thiol- metallo- and mixed proteases. These results further underscore the significance of these enzymes in functionally facilitating parasite survival within their infected hosts. In parallel, we also demonstrated that promastigotes of all pathogenic Leishmania species released very high levels of a soluble, secretory isoform of a zinc-metallo-protease enzyme (GP-63S). Our results suggest that this secretory protease helps facilitate parasite growth in its various host environments. In other biochemical and molecular studies, we demonstrated that a unique L. donovani ARF1-protein functions to maintain the structural integrity of the trans-Golgi cisternal network and facilitates the proper trafficking and transit of both secretory and surface membrane proteins through the Golgi compartment in these parasites. Further, using site specific targeted-mutations, we showed that the LdARF1-protein was essential for the survival of these organisms. In addition to the foregoing, recently we succeeded in developing an in vitro system for generating and propagating large quantities of infectious forms (i.e. axenic amastigotes) of L. donovani. The availability of such material should now greatly facilitate studies in general concerning the basic cell and molecular biology of this human parasite. Cumulatively, the results of our recent and ongoing studies continue to provide pertinent and significant information toward understanding the unique pathophysiology of these parasites. In addition, these studies are of practical relevance toward demonstrating whether specific /unique parasite enzymes and regulatory proteins are logical targets for 1) the design of new chemotherapeutic drugs, 2) the development of new diagnostic tools and/or 3) useful as potential vaccines against these human pathogens.